CN111551658A - Rapid detection and analysis method of antihistamines in water - Google Patents

Abstract

The invention discloses a rapid detection and analysis method for antihistamine drugs in water, which can be used for the detection of drugs for treating allergic diseases. At the same time, 6 kinds of antihistamines were detected in water, including cimetidine, diphenhydramine, cetirizine, fexofenadine, chlorpheniramine, and ranitidine. The method includes the following steps: firstly filter the water sample and add internal standard substance; then use a solid phase extraction cartridge to extract and purify 6 kinds of statins in the sample; and then detect the water sample by high performance liquid chromatography-mass spectrometer target content. The invention has simple processing steps for water samples, convenient operation and good stability, and can quickly obtain samples suitable for detection by liquid mass spectrometer; the sample pretreatment cost is low, can be completed by using common consumables in laboratories, and the detection speed is fast , high degree of automation, sensitive response, suitable for popularization and application.

Description

Rapid detection and analysis method of antihistamines in water

technical field

The invention relates to a detection method for drugs for treating allergic diseases, in particular to a detection and analysis method for antihistamine drugs, which is applied to the technical field of trace detection of organic pollutants in water bodies.

Background technique

With the progress and development of modern medicine, a large number of drugs are produced and consumed every year in the world for the prevention and treatment of human and animal diseases. These drugs enter natural water bodies and water bodies through a variety of routes. The most common way is the discharge of industrial sewage near pharmaceutical factories and the discharge of domestic sewage caused by the metabolism of human and animal bodies. After the previous literature research, it has been detected that there are certain concentrations of drugs and their metabolites in sewage and the environment. Although their concentration levels are several orders of magnitude lower than the specified toxic risk concentrations for humans and animals, their effects on the environment The long-term impact cannot be ignored.

As a common class of drugs, antihistamines specifically function to antagonize the biological effects of histamine on the human body, thereby avoiding local congestion caused by histamine-induced telangiectasia and increased permeability, smooth muscle spasm, and enhanced secretion activity. , edema, increased secretions, bronchial and digestive tract smooth muscle contraction and other clinical symptoms. Antihistamines are widely used in clinical practice due to their easy preparation, low price, convenient administration, and effectiveness against various allergic diseases.

Antihistamines are continuously released into water bodies through various channels. Due to the poor removal efficiency, even the water treated by drinking water treatment plants will contain statins, which will adversely affect humans and organisms. Due to the complex composition of water samples and the presence of organic and inorganic pollutants, the existence of these substances greatly interferes with accurate and rapid detection, and requires high levels of purification and enrichment of samples. At present, the pretreatment methods for water samples are mainly liquid-liquid extraction and glass rod extraction, which consume a lot of extraction solvent, require a long time for experiments, and have complicated steps. This has become an urgent technical problem to be solved.

SUMMARY OF THE INVENTION

In order to solve the problems of the prior art, the purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a rapid detection and analysis method for antihistamines in water bodies, which can pretreat environmental water bodies to improve the existing treatment methods. The process is complicated and the cost is high. At the same time, the present invention adopts a high-performance liquid chromatography-mass spectrometry detection method for antihistamine drugs, and the detection method can simultaneously detect 6 kinds of statin drug substances, including cimetidine, diphenhydramine, cetirizine, Fexofenadine, chlorpheniramine, ranitidine, the detection process is fast, efficient, low-cost, highly automated and easy for industrial application.

In order to achieve the above-mentioned purpose of invention and creation, the present invention adopts the following technical solutions:

A method for rapid detection and analysis of antihistamines in water, comprising the following steps:

a. Pretreatment of samples:

Add internal standard cetirizine-d8 and diphenhydramine-d3 to water samples and mix well;

b. Use solid phase extraction column for enrichment and purification:

The specific process of passing the column is as follows: firstly, activate the solid-phase extraction column with ultrapure water or methanol of no more than 6mL, and then pass the filtered 200mL water sample through the chromatographic column at a speed of 2ml/min, and then use no more than 5mL ultrapure water to pass through the column. Rinse with pure water at a flow rate of 1 to 3 mL/min, then dry at a pressure of 0.6 bar for at least 30 min, and finally elute with no more than 6 mL of methanol; after blowing to near dryness in a nitrogen atmosphere at 40 °C, use 1 mL of methanol to dilute to volume , and then transferred to HPLC vials for detection;

c. Determination of the content of the target substance in the sample by high performance liquid chromatography-mass spectrometry:

A standard curve of the target pollutants was established, with the concentration as the abscissa and the peak area as the ordinate; the internal standard method was used to quantitatively detect the concentration of the target compounds extracted from the water samples on a high performance liquid chromatography-mass spectrometer.

As a preferred technical solution of the present invention, in the step a, the sample is roughly filtered at least three times and finely filtered at least three times; the total concentration of the internal standard added in each 250 mL of water sample is 20-100 ppb.

As a preferred technical solution of the present invention, in the step b, the process of drying with nitrogen is 45-60 min.

As a preferred technical solution of the present invention, in the step c, the detection conditions of the high performance liquid chromatography-mass spectrometer are:

The column temperature was 35 °C, the injection volume was 3 μL, and the flow rate was 0.3 mL/min;

The mobile phase adopts HPLC grade ammonium formate-formic acid solution (A) and acetonitrile solution of formic acid (B), wherein the ammonium formate concentration in the HPLC grade ammonium formate-formic acid solution (A) is 0.1mol/L, and the HPLC grade ammonium formate-formic acid The pH of the solution (A) is 3.5, and the formic acid concentration in the formic acid in acetonitrile solution (B) is 0.1 mol/L; the gradient (wt.% A) is as follows: 0min, 95%; 1min, 95%; 3min, 80%; 5min , 80%; 5.01min, 65%; 9min, 55%; 9.01min, 0%; 11min, 0%; 11.0min, 95%; 12min, 95%;

Drying gas flow rate: 8L/min, drying gas temperature: 300℃, sheath gas temperature: 350℃, sheath gas flow rate: 11L/min, atomizer pressure: 60psi, nozzle voltage (-): 1500V, nozzle voltage (+ ): 1500V, capillary voltage (-): 4500.

As a preferred technical solution of the present invention, in the step c, the concentration of cimetidine, diphenhydramine, cetirizine, fexofenadine, chlorpheniramine and ranitidine in the water sample is carried out. detection.

Compared with the prior art, the present invention has the following obvious outstanding substantive features and significant advantages:

1. The present invention establishes a pretreatment method of membrane filtration and solid-phase extraction in series, and then uses a solid-phase extraction small column in series with a high-performance liquid chromatography mass spectrometry method to scan under the modes of primary mass spectrometry and secondary mass spectrometry, respectively. The advantages of high stability, good repeatability and accurate results;

2. The pretreatment method of the present invention is simple, easy to operate, and the selected instrument has good resolution and high accuracy. After multi-level scanning, the sensitivity and reliability of the method are enhanced, and the results of data analysis and processing are simple to present. It is clear that the qualitative analysis is comprehensive and comprehensive, and the function of comprehensive screening of non-target pollutants can be realized;

3. The intra-day precision and inter-day precision of the method of the present invention both meet the requirements, and the relative standard deviations (RSDs) are respectively less than 9.8%. The method has good precision, high repeatability, accurate, sensitive, and reliable results, and has a wide range of applications.

Description of drawings

Fig. 1 is the chromatogram of six kinds of antihistamines detected by the method of the second embodiment of the present invention.

Detailed ways

The above scheme will be further described below in conjunction with specific embodiments, and preferred embodiments of the present invention are described in detail as follows:

Example 1:

In the present embodiment, a method for rapid detection and analysis of antihistamines in water, the standard addition recovery experiment, using the internal standard method to detect the recovery of antihistamines in natural water, includes the following steps:

a. Pretreatment of samples:

The water samples were coarsely filtered three times and finely filtered three times; three groups of parallel samples were set up, each group of water samples was taken 250ml, and the total concentration of 20ppb of cetirizine-d8 and diphenhydramine-d3 solutions was added to each group of water samples. 1mL, mix well;

b. Use solid phase extraction column for enrichment and purification:

The specific process of passing the column is: firstly activate the solid-phase extraction column with 5mL of methanol, secondly pass the filtered 200mL water sample through the chromatographic column at a speed of 2ml/min, and then use 5mL of ultrapure water at a flow rate of 3mL/min. Rinse, then dry at 0.6 bar for 30 min, and finally elute with 3 ml of methanol; after blowing to near dryness in a nitrogen atmosphere at 40 °C, make up to volume with 1 mL of methanol, and then transfer to a high performance liquid chromatography bottle for detection ;

c. Determination of the content of the target substance in the sample by high performance liquid chromatography-mass spectrometry:

A standard curve of the target pollutants was established, with the concentration as the abscissa and the peak area as the ordinate; the internal standard method was used to quantitatively detect the concentration of the target compounds extracted from the water samples on a high performance liquid chromatography-mass spectrometer.

The detection conditions of high performance liquid chromatography-mass spectrometer are:

The column temperature was 35 °C, the injection volume was 3 μL, and the flow rate was 0.3 mL/min;

The mobile phase adopts HPLC grade ammonium formate-formic acid solution (A) and acetonitrile solution of formic acid (B), wherein the ammonium formate concentration in the HPLC grade ammonium formate-formic acid solution (A) is 0.1mol/L, and the HPLC grade ammonium formate-formic acid The pH of the solution (A) is 3.5, and the formic acid concentration in the formic acid in acetonitrile solution (B) is 0.1 mol/L; the gradient (wt.% A) is as follows: 0min, 95%; 1min, 95%; 3min, 80%; 5min , 80%; 5.01min, 65%; 9min, 55%; 9.01min, 0%; 11min, 0%; 11.0min, 95%; 12min, 95%;

Drying gas flow rate: 8L/min, drying gas temperature: 300℃, sheath gas temperature: 350℃, sheath gas flow rate: 11L/min, atomizer pressure: 60psi, nozzle voltage (-): 1500V, nozzle voltage (+ ): 1500V, capillary voltage (-): 4500.

Experimental test analysis:

In this embodiment, the standard addition recovery rate is used for calculation. The calculation formula of the recovery rate of standard addition is: RE%=(C ₂ V ₂ -C ₁ V ₁ )/C ₀ V ₀ where: RE: recovery rate of standard addition, %; C ₀ : concentration of mixed standard solution, ng/ mL; V ₀ : volume of mixed standard solution, mL; C ₁ : detection concentration of blank sample, ng/mL; V ₁ : volume of blank sample at constant volume, mL; C ₂ : detection of sample added with mixed standard solution Concentration ng/mL; V ₂ : the volume of the sample added to the mixed standard solution at constant volume, mL.

The measured result in this example is 58.56 μg/L, the recovery rate of standard addition is 79.62%, and the relative standard deviation (n=3) is 4.98. It can be seen that the detection accuracy of this method is better.

The test results can be corrected through the standard addition recovery experiment to eliminate human error and reduce the effect of matrix interference. The solid-phase small column extraction method adopted in the present invention can effectively separate the components to be tested and the sample matrix, overcome the matrix effect, have good reproducibility of experimental results, high recovery rate and accurate detection results.

Embodiment 2:

This embodiment is basically the same as the first embodiment, and the special features are:

In this example, the concentration of antihistamines in actual samples is measured, and 5 water samples in the environment are randomly collected, pretreated with the method of step a of Example 1, and then the solid phase extraction method of step b of Example 1 is used. Purification and enrichment are carried out, and then the method of high performance liquid chromatography-mass spectrometry in step c of Example 1 is used to analyze and detect the actual concentration of the target substance in the sample. The specific detection process is as follows:

In the present embodiment, a method for rapid detection and analysis of antihistamines in water, the standard addition recovery experiment, using the internal standard method to detect the recovery of antihistamines in natural water, includes the following steps:

a. Pretreatment of samples:

The water samples were coarsely filtered three times and finely filtered three times; three groups of parallel samples were set up, each group of water samples was taken 250ml, and the total concentration of 20ppb of cetirizine-d8 and diphenhydramine-d3 solutions was added to each group of water samples. 1mL, mix well;

b. Use solid phase extraction column for enrichment and purification:

The specific process of passing the column is: firstly activate the solid-phase extraction column with 5mL of methanol, secondly pass the filtered 200mL water sample through the chromatographic column at a speed of 2ml/min, and then use 5mL of ultrapure water at a flow rate of 3mL/min. Rinse, then dry at 0.6 bar for 30 min, and finally elute with 3 ml of methanol; after blowing to near dryness in a nitrogen atmosphere at 40 °C, make up to volume with 1 mL of methanol, and then transfer to a high performance liquid chromatography bottle for detection ;

c. Determination of the content of the target substance in the sample by high performance liquid chromatography-mass spectrometry:

A standard curve of the target pollutants was established, with the concentration as the abscissa and the peak area as the ordinate; the internal standard method was used to quantitatively detect the concentration of the target compounds extracted from the water samples on a high performance liquid chromatography-mass spectrometer.

The detection conditions of high performance liquid chromatography-mass spectrometer are:

The column temperature was 35 °C, the injection volume was 3 μL, and the flow rate was 0.3 mL/min;

The mobile phase adopts HPLC grade ammonium formate-formic acid solution (A) and acetonitrile solution of formic acid (B), wherein the ammonium formate concentration in the HPLC grade ammonium formate-formic acid solution (A) is 0.1mol/L, and the HPLC grade ammonium formate-formic acid The pH of the solution (A) is 3.5, and the formic acid concentration in the formic acid in acetonitrile solution (B) is 0.1 mol/L; the gradient (wt.% A) is as follows: 0min, 95%; 1min, 95%; 3min, 80%; 5min , 80%; 5.01min, 65%; 9min, 55%; 9.01min, 0%; 11min, 0%; 11.0min, 95%; 12min, 95%;

Drying gas flow rate: 8L/min, drying gas temperature: 300℃, sheath gas temperature: 350℃, sheath gas flow rate: 11L/min, atomizer pressure: 60psi, nozzle voltage (-): 1500V, nozzle voltage (+ ): 1500V, capillary voltage (-): 4500.

Experimental test analysis:

The concentrations of antihistamines in the 5 water samples detected in this example are shown in Table 1 below. The experimental results show that the method of this embodiment can be applied to the determination of antihistamines in environmental water samples.

Table 1. Concentration test results of 6 antihistamines in water samples

The solid-phase extraction cartridge in step b of this example is an Oasis HLB-type cartridge from Waters. In step c of this example, the model of the high-performance liquid chromatography-mass spectrometer is a SCIEX QTRAP 5500 liquid chromatograph, and the chromatographic column is a Kinetex120EC-C18 reversed-phase column (3×100 mm, 2.7 μm, phenomenex). This embodiment utilizes solid phase extraction cartridges combined with high performance liquid chromatography-mass spectrometry technology to efficiently and rapidly detect 6 kinds of antihistamines in water, including cimetidine, diphenhydramine, and cetirizine. , Fexofenadine, Chlorpheniramine, Ranitidine, etc. Fig. 1 is the chromatogram of six kinds of antihistamines detected by the method of this embodiment. It can be seen from Figure 1 that the chromatographic peaks of the antihistamines obtained by this method are beautiful, and the separation effect is good, and the signal intensities of various drugs are very high, all above 10e5. Table 1 shows the effects of the solid-phase small column extraction method used in this example. The method tests the recovery rates of six antihistamines in five water samples, and the recovery rates are all above 80%, showing a higher The recovery rate shows that the method can effectively separate the components to be tested and the sample matrix, and ensure accurate detection results.

To sum up, the above embodiments of the present invention are used for the detection method of drugs for the treatment of allergic diseases, using solid phase extraction cartridges combined with high performance liquid chromatography-mass spectrometry technology, can efficiently and rapidly detect 6 kinds of antihistamines in water bodies at the same time. Class of drugs, including cimetidine, diphenhydramine, cetirizine, fexofenadine, chlorpheniramine, ranitidine. The method comprises the following steps: (1) filtering the water sample and adding an internal standard substance; (2) extracting and purifying 6 kinds of statins in the sample by using a solid phase extraction cartridge; (3) passing the high performance liquid chromatography- The mass spectrometer detects the content of the target substance in the water sample. The invention has the advantages of simple processing steps for water samples, convenient operation and good stability, and can quickly obtain samples suitable for detection by liquid mass spectrometer; the sample pretreatment cost is low, can be completed by using common consumables in the laboratory, and can be used in various laboratories. The pre-class processing lab is completed. This method can simultaneously detect 6 kinds of antihistamines quickly and efficiently, with fast detection speed, high degree of automation, sensitive response, and the recovery rate can reach 70.62-110.37%, which is convenient for industrial application. Fast and accurate qualitative and quantitative detection method, suitable for popularization and application.

The embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned embodiments, and various changes can also be made according to the purpose of the invention and creation of the present invention. The changes, modifications, substitutions, combinations or simplifications should be equivalent substitution methods, as long as they meet the purpose of the invention, as long as they do not deviate from the technical principles and inventions of the rapid detection and analysis method for antihistamines in water of the invention All ideas belong to the protection scope of the present invention.

Claims (5)

1. A method for rapidly detecting and analyzing antihistamine medicines in a water body is characterized by comprising the following steps:

a. pretreatment of the sample:

adding internal standard substance cetirizine-d 8 and diphenhydramine-d 3 into a water body sample, and uniformly mixing;

b. enrichment and purification by using a solid phase extraction column:

the specific flow of column passing is as follows: firstly activating a solid phase extraction column by using ultrapure water or methanol of not more than 6mL, secondly enabling a filtered 200mL water sample to pass through a chromatographic column at the speed of 2mL/min, then rinsing the column by using ultrapure water of not more than 5mL at the flow rate of 1-3 mL/min, then drying the column for at least 30min under the air pressure of 0.6bar, and finally eluting the column by using methanol of not more than 6 mL; after being blown to be nearly dry in the nitrogen atmosphere at the temperature of 40 ℃, the volume is determined by 1mL of methanol, and then the solution is transferred to a high performance liquid chromatography bottle for detection;

c. determining the content of a target object in a sample by using a high performance liquid chromatography-mass spectrometer:

establishing a standard curve of the target pollutant, taking the concentration as a horizontal coordinate and taking a peak area as a vertical coordinate; and (3) quantitatively detecting the concentration of the target object extracted from the water sample on a high performance liquid chromatography-mass spectrometer by adopting an internal standard method.

2. The method for rapid detection and analysis of antihistamines in a body of water according to claim 1, wherein: in the step a, the sample is coarsely filtered for at least three times, and finely filtered for at least three times; the total concentration of the added internal standard substance in each 250mL of water body sample is 20-100 ppb.

3. The method for rapid detection and analysis of antihistamines in a body of water according to claim 1, wherein: in the step b, the process of drying by nitrogen is 45-60 min.

4. The method for rapid detection and analysis of antihistamines in a body of water according to claim 1, wherein: in the step c, the detection conditions of the high performance liquid chromatography-mass spectrometer are as follows:

the column temperature is 35 ℃, the sample injection volume is 3 mu L, and the flow rate is 0.3 mL/min;

the mobile phase adopts HPLC-grade ammonium formate-formic acid solution (A) and acetonitrile solution (B) of formic acid, wherein the concentration of ammonium formate in the HPLC-grade ammonium formate-formic acid solution (A) is 0.1mol/L, the pH of the HPLC-grade ammonium formate-formic acid solution (A) is 3.5, and the concentration of formic acid in the acetonitrile solution (B) of formic acid is 0.1 mol/L; gradient (wt.% a) as follows: 0min, 95%; 1min, 95%; 3min, 80%; 5min, 80%; 5.01min, 65%; 9min, 55%; 9.01min, 0%; 11min, 0%; 11.0min, 95%; 12min, 95%;

flow rate of drying gas: 8L/min, drying gas temperature: 300 ℃, sheath gas temperature: 350 ℃, sheath gas flow: 11L/min, atomizer pressure: 60psi, nozzle voltage (-): 1500V, nozzle voltage (+): 1500V, capillary Voltage (-): 4500.

5. the method for rapid detection and analysis of antihistamines in a body of water according to claim 1, wherein: in the step c, the concentration of cimetidine, diphenhydramine, cetirizine, fexofenadine, chlorpheniramine and ranitidine in the water body sample is detected.

Images